Colorectal cancer (CRC) is one of the most common human malignancies and is second in cancer- related death, and is even more prevalent in the developed countries including the US. Genetic heterogeneity of CRCs renders it a major therapeutic challenge. New therapy is urgently needed to improve the mortality of CRC patients. Target of rapamycin (TOR) is a conserved protein kinase and a key regulator of cell growth and survival, acting downstream of PI3K. PI3K-mammalian/mechanistic TOR (mTOR) pathway is frequently hyper-activated in human cancers, leading to uncontrolled cancer growth, which is a major cancer drug target. mTOR forms two distinct multimeric protein complexes, mTORC1 and mTORC2, both of which are required for cancer cell growth, proliferation and survival. The macrolide rapamycin and rapamycin analogs (rapalogs) are partial mTOR inhibitors with limited efficacy toward major human cancers. Recently, mTOR kinase inhibitors (mTKIs) have emerged as the second generation of mTOR-targeted therapeutics. Early studies showed that mTKIs are indeed effective against several rapamycin-resistant tumor models. As a result, a large number of mTKIs rapidly entered human clinical trials. The extraordinary speed from initial drug discovery to human clinical trials underscores the therapeutic potential of this class of new anti-neoplastic agents. Despite early progress, significant challenges remain. Thus far there have been few studies on their mechanisms of action. Moreover, there has been no reported study on intrinsic or acquired resistance to this new drug class. Our application is aimed at answering these critically important yet unaddressed questions. The rationale and feasibility of our research plan are strongly supported by the preliminary results. Successful completion of this project should have significant impact on the clinical development of this new class of anti-neoplastic agents. PUBLIC HEALTH RELEVANCE: Colorectal cancer (CRC) is one of the most common human malignancies and is second in cancer- related death, and is even more prevalent in the developed countries including the US. Genetic heterogeneity of CRCs renders it a major therapeutic challenge. New therapy is urgently needed to improve the mortality of CRC patients. Target of rapamycin (TOR) is a conserved PI3K-related kinase and a key regulator of cell growth and survival. Mammalian TOR (mTOR) pathway is frequently hyper- activated in human cancer such as CRC, leading to uncontrolled cancer growth and proliferation, rendering TOR as a highly desirable cancer drug target. The macrolide rapamycin and rapamycin analogs (rapalogs) are used in the clinic to treat several different human diseases, including advanced renal cell carcinomas. However, rapalogs are only partial mTOR inhibitors, and except for renal cancer and several other rare tumors, are not very effective against major human cancers. Recently, a second generation of anti-mTOR drugs called mTOR kinase inhibitors (mTKIs) has been developed. Preclinical studies show that they are indeed more potent anti-cancer agents than rapalogs. As a result, a large number of mTKI compounds have rapidly entered human clinical trials. The extraordinary speed from initial drug discovery to human clinical trials underscores their therapeutic potential. Despite early progress, significant challenges remain. Thus far the effect of mTKIs toward CRCs has not been explored and the understanding of their mechanisms of action is lacking. Moreover, there is virtually no research on drug resistance to these drugs, which is critically important for clinical development of these drugs. This applicatio will address these critically important questions. Successful completion of this project should have significant impact on the clinical development of this new class of anti- neoplastic agents, and improve the treatment of colorectal cancer.